KR102139203B1 - AAV-Vectors for use in gene therapy of choroideremia - Google Patents

Abstract

The present invention relates to gene therapy for the treatment or prevention of pancreatic atrophy.

Description

AAV-Vectors for use in gene therapy of choroideremia}

The present invention relates to gene therapy for the treatment or prevention of pancreatic atrophy.

Pancreatic atrophy is a rare X chromosome-associated progressive degeneration of the choroid of the eye, retinal pigment epithelium, and photoreceptors. A typical natural history in male patients is night blindness in teenagers, followed by loss of peripheral vision in their 20s and 30s, leading to complete blindness in their 40s. Female carriers have mild symptoms, most notably night blindness, but sometimes they may have a more severe phenotype.

The disease is caused by a mutation in the REP1 gene (Rab escort protein 1) located in the 21 chromosome X region. In most cells in the body, the REP2 protein, which is 75% homologous to REP1, compensates for REP1 deficiency. However, in the eye, for reasons not yet clear, REP2 does not compensate for REP1 deficiency. Thus, in the eye, REP polypeptide activity is insufficient to maintain normal prenylation of the target protein (Rab GTPase), leading to cell dysfunction and ultimately cell death, which mainly affects the external retina and choroid. .

There is no treatment for pancreatic atrophy, and there is a lack of a model for evaluating treatment strategies. There is a need to provide such treatment.

Summary of the invention

The present invention relates to a vector that can be used for gene therapy of pancreatic atrophy, and a method for preventing or treating the disease using the vector. The present invention also relates to the use of a vector in a method of preventing or treating pancreatic atrophy.

The vector of the present invention is a viral vector, specifically a viral vector based on the genome of adeno-associated virus (AAV). This vector contains a sequence encoding REP1 or a variant thereof, thus allowing REP1 function to be expressed in the target cell. The methods and uses of the present invention specifically include administering a vector to a patient by direct retinal injection, subretinal injection, or intravitreal injection for the treatment or prevention of pancreatic atrophy. Includes.

Accordingly, the present invention provides a vector comprising a polynucleotide sequence encoding an adeno-associated virus (AAV) genome or a derivative thereof and REP1 or a variant thereof. The present invention also administers a therapeutically effective amount of a vector according to any one of the preceding claims by direct retinal injection, subretinal injection, or intravitreal injection, to a patient in need of treatment or prevention of pancreatic atrophy, whereby said It provides a method of treating or preventing pancreatic atrophy, comprising the step of treating or preventing pancreatic atrophy in a patient. The present invention further provides a vector of the present invention for use in a method of treating or preventing pancreatic atrophy by administering the vector of the present invention to a patient by direct retinal injection, subretinal injection, or intravitreal injection.

Brief description of the drawing

Figure 1 shows that the AAV.REP1 (AAV-CAG-REP1) vector can be efficiently transduced into human fibroblasts isolated from patients with pancreatic atrophy (Chm). The relative level of expression of the human REP1 protein (hREP1) is compared to the amount of hREP1 in different concentrations of cell lysate to quantify the activity of the AAV2.REP1 vector in Western blot. By comparison. With regard to labeling, CAG is Chicken beta Actin with CMV enhancer promoter sequence-may be referred to as'CBA' compatible in various publications and parts of this specification. .

Western blots are indicated in the left panel for the loading control REP1 (top panel) and alpha-tubulin (bottom panel). Lane 1: 40 μg cell lysate of control wild type (WT) fibroblasts. Lane 2: 40 μg cell lysate of Chm fibroblasts. Lanes 3-6: 40, 20, 10 and 5 μg cell lysates of Chm fibroblasts transduced with AAV2.REP1 vector. Lane 7: human REP1 recombinant protein. Since the 5 μg lysate hREP1 band has a density similar to that of the 40 μg WT fibroblast lysate, the level of hREP1 achieved by the AAV2.REP1 vector achieves at least 8 times (40/5) the normal wild-type level under these conditions. can do.

As a positive control for promoters and other non-REP1 sequences in this assay, the results of the control AAV vector (AAV-CAG-GFP) expressing green fluorescent protein (GFP) instead of REP1 are also shown.

Western blots are shown in the right panel for the loading control GFP (top panel) and alpha-tubulin (bottom panel). Lane 1: 40 μg cell lysate of wild type (WT) fibroblasts. Lane 2: 40 μg cell lysate of Chm fibroblasts (AAV-CAG-REP1) transduced with AAV2.GFP1 vector. The high level of GFP indicated confirms the efficiency of this vector expression cassette in transduction of human cells deficient in REP1 activity, as in the case of pancreatic atrophy.

Figure 2 WT human fibroblasts (first column from left (column)-light gray), Chm fibroblasts (second column-dark gray), AAV-CAG-GFP vector transduced Chm fibroblasts (third column-white) , Negative control) and Chm fibroblasts transduced with AAV-CAG-REP1 (fourth column-white). The Y axis shows the amount of radiolabeled substrate [3H] GGPP substrate delivered in pmol, which is a measure of prenylation, REP1 function. Columns show error bars as standard deviation (n=4 for each column). The level of [3H]-GGPP was measured in 10 mg of protein extract. The fourth turquoise column from the left confirms that the function of prenylation is restored to wild-type levels and higher after transduction of the AAV.REP1 vector. This confirms that the REP1 protein detected by Western blot in FIG. 1 has the expected function.

FIG. 3 shows that the AAV vector has accurate tropism for outer retinal (photoreceptor and choroid) cells after subretinal injection in a mouse model. The right panel shows the appropriateness of the green fluorescent protein (GFP) marker (arrow) in the photoreceptors of the outer nuclear layer (ONL) and retinal pigment epithelium (RPE) after subretinal injection of the AAV2.CBA.GFP.WPRE.BGH vector from the mouse eye Show expression. The left panel shows the grayscale of the same image. This demonstrates that the AAV2.CBA.WPRE.BGH regulatory sequence can express the transgene with high efficiency in retinal cells that should be targeted in patients with pancreatic atrophy.

4 shows that the AAV.REP1 vector does not negatively affect the outer retina function of the mouse retina at high doses. Toxic research results of measuring retinal potential (ERG) for 6 months after 2 x 1 microliter subretinal injection of a high dose (n=5) or low dose (n=4) AAV.REP1 vector into the subretinal space of a mouse are displayed do. Low dose is 1 x 10 ¹¹ genome particles per ml (gp) and high dose is 1 x 10 ¹² gp per ml (the starting dose for human clinical trials is 1 x 10 ¹¹ gp per ml). AAV.GFP vectors have the same expression cassette and are also diluted to the same dose prior to injection to serve as a control. The Y-axis shows ERG traces at increasing scintillation intensity in the range of brightness above the dark adaptation (dark adaptation) rod response and below the light adaptation response (including the photoreceptors of the abstract body). Records at all points show similar ERG amplitudes after high and low dose AAV.REP1 exposure. In the high-dose group, the corresponding GFP amplitude is slightly reduced, consistent with a known marginal effect on the retinal function of GFP at high levels. This GFP effect also serves as a positive control to confirm the sensitivity of this test.

Description of the sequence

SEQ ID NO: 1 is the DNA sequence for the AAV2 genome.

SEQ ID NO: 2 is a DNA sequence encoding the human Rep-1 protein, transcript variant 1.

SEQ ID NO: 3 is the amino acid sequence for human Rep-1 protein, transcription variant 1.

SEQ ID NO: 4 is a DNA sequence encoding the human Rep-1 protein, transcription variant 1, including a portion of the 5'UTR.

SEQ ID NO: 5 is the DNA sequence for the woodchuck hepatitis postregulatory element (WPRE).

SEQ ID NO: 6 is the DNA sequence for the chicken beta actin (CBA) promoter.

SEQ ID NO: 7 is the DNA sequence for the polyadenylation site (bGH polyA) of bovine growth hormone.

SEQ ID NO: 8 is the DNA sequence for the 5'inverted terminal repeat (ITR) of AAV2.

SEQ ID NO: 9 is the DNA sequence for 3'ITR of AAV2.

Detailed description of the invention

The present invention provides a method of treating pancreatic atrophy. It is based on a gene therapy approach to pancreatic atrophy, using a genetic construct that delivers the transgene to restore REP1 function. The genetic construct is a vector based on an adeno-associated virus (AAV) genome comprising a polynucleotide sequence encoding REP1 or a variant thereof. This polynucleotide sequence is also referred to herein as a “transgene”. The inventors have established a model to evaluate strategies for treatment of pancreatic atrophy, and surprisingly demonstrated the use of the vector of the invention to target cell dysfunction inherent in pancreatic atrophy.

vector

AAV Genome

The vector of the present invention first comprises an adeno-associated virus (AAV) genome or a derivative thereof.

The AAV genome is a polynucleotide sequence that encodes the functions necessary for the production of AAV virus particles. These functions include those that operate in the replication and packaging cycle for AAV in host cells, including encapsidation of the AAV genome into AAV virus particles. Natural AAV virus lacking the ability to replicate and depends on the provision of helper function in trans (trans) to the completion of the replication and packaging cycle. Therefore, the AAV genome of the vector of the present invention generally lacks the ability to replicate.

The AAV genome can be positive or negative-sense, single-stranded or double-stranded. The use of the double-stranded form enables bypass of the DNA replication step in the target cell, thus accelerating the expression of the transgene.

The AAV genome can be derived from a naturally-derived serotype or isolate or clade of AAV. Thus, the AAV genome can be the entire genome of a native AAV virus. As known to those skilled in the art, naturally occurring AAV viruses can be classified according to various biological systems.

Generally, AAV viruses are referred to according to their serotype. Serotypes correspond to variant subspecies of AAV with unique reactivity that can be used to differentiate from other variant subspecies due to the expression profile of their capsid surface antigen. In general, viruses having a specific AAV serotype do not efficiently cross-react with neutralizing antibodies specific for other AAV serotypes. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, and also include recombinant serotypes such as Rec2 and Rec3, recently identified in the primate brain.

The preferred serotype of AAV for use in the present invention is AAV2. The AAV2 genome may have the sequence of SEQ ID NO: 1. Other serotypes of particular interest for use in the present invention include AAV4, AAV5 and AAV8 that are efficiently transduced into intraocular tissues such as retinal pigment epithelium. The serotype of AAV used may be an AAV serotype rather than AAV4. Review of AAV serotype by Choi et al. ( Curr Gene Ther . 2005; 5(3); 299-310) and Wu et al. ( Molecular Therapy ; 14(3), 316-327). The sequence of the AAV genome sequence or the element of the AAV genome, including the ITR sequence, rep or cap gene used in the present invention can be derived from the accession number of the following AAV full genome sequence: adeno- Related virus 1 NC_002077, AF063497; Adeno-associated virus 2 NC_001401; Adero-associated virus 3 NC_001729; Adeno-associated virus 3B NC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Algae AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889, AY388617.

AAV virus is also referred to in terms of lineages or clones. It represents the phylogenetic relationship of naturally-derived AAV viruses, and the phylogenetic group of AAV viruses that can generally be traced back to a common ancestor, including all their progeny. In addition, the AAV virus may also be referred to according to a specific isolate, such as the genetic isolate of a specific AAV virus found in nature. The term genetic isolate refers to a population of AAV viruses, where limited genetic mixing occurs with other native AAV viruses, thereby defining a distinct population that is recognibly distinct at the genetic level. Describe.

Examples of phylogenetic groups and isolates of AAV that can be used in the present invention include:

Lineage A: AAV1 NC_002077, AF063497, AAV6 NC_001862, Hu. 48 AY530611, Hu 43 AY530606, Hu 44 AY530607, Hu 46 AY530609

Families B: Hu. 19 AY530584, Hu. 20 AY530586, Hu 23 AY530589, Hu22 AY530588, Hu24 AY530590, Hu21 AY530587, Hu27 AY530592, Hu28 AY530593, Hu 29 AY530594, Hu63 AY530624, Hu64 AY530625, Hu13 AY530578, Hu56 AY530618, Hu57 AY530619530, Huya AY530619530 Huya Hu35 AY530599, AAV2 NC_001401, Hu45 AY530608, Hu47 AY530610, Hu51 AY530613, Hu52 AY530614, Hu T41 AY695378, Hu S17 AY695376, Hu T88 AY695375, Hu T71 AY695374, Hu T70 AY695373, Hu T40 AY6937, Huy T40 AY69537 Hu LG15 AY695377,

Families C: Hu9 AY530629, Hu10 AY530576, Hu11 AY530577, Hu53 AY530615, Hu55 AY530617, Hu54 AY530616, Hu7 AY530628, Hu18 AY530583, Hu15 AY530580, Hu16 AY530581, Hu25 AY530591, Hu60 AY530622, Ch5 AY530021, Ch5 AY AY530602 Hu2, AY530585, Hu61 AY530623

Lineage D: Rh62 AY530573, Rh48 AY530561, Rh54 AY530567, Rh55 AY530568, Cy2 AY243020, AAV7 AF513851, Rh35 AY243000, Rh37 AY242998, Rh36 AY242999, Cy6 AY243016, Cy4 AY243018, Cy3 AY13430243

Families E: Rh38 AY530558, Hu66 AY530626, Hu42 AY530605, Hu67 AY530627, Hu40 AY530603, Hu41 AY530604, Hu37 AY530600, Rh40 AY530559, Rh2 AY243007, Bb1 AY243023, Bb2 AY243022, Rh10 AY243015, Hu530 A5357 AY530554, Pi1 AY530553, Pi3 AY530555, Rh57 AY530569, Rh50 AY530563, Rh49 AY530562, Hu39 AY530601, Rh58 AY530570, Rh61 AY530572, Rh52 AY530565, Rh53 AY530566, Rh51 AY530564, Rh64 A530

Lineage F: Hu14 (AAV9) AY530579, Hu31 AY530596, Hu32 AY530597, Clonal Isolate AAV5 Y18065, AF085716, AAV 3 NC_001729, AAV 3B NC_001863, AAV4 NC_001829, Rh34 AY243001, Rh33 AY243002, Rh33 AY243002,

Those skilled in the art can select appropriate serotypes, lineages, clones or isolates of AAV for use in the present invention based on their common general knowledge. For example, AAV5 capsids have been found to be efficiently transduced into primate abstract photoreceptors (mancuso et al., Nature 2009, 461:784), as evidenced by successful correction of genetic color vision defects. -7).

However, it should be understood that the present invention also encompasses the use of other serotype AAV genomes that may not have been identified or identified yet. The AAV serotype determines the tissue specificity of the infection (or host activity) of the AAV virus. Therefore, the preferred AAV serotype for use in the AAV virus administered to a patient according to the present invention is a serotype having a natural migration activity against target cells in degenerated retina of pancreatic atrophy or high infection efficiency. Thus, the preferred AAV serotype for use in the AAV virus administered to patients is the AAV serotype that infects cells of the retinal optic epithelium and the neurons of the retina.

Generally, the naturally occurring serotype or isolate of AAV or the AAV genome of a lineage includes one or more inverted terminal repeat sequences (ITRs). ITR sequences serve to provide a functional origin of replication in the cis (cis) position, and allows the fusion and cutting from it the vector from the genome of the cell. In a preferred embodiment, the one or more ITR sequences flank the polynucleotide sequence encoding Rep-1 or variant thereof. A preferred ITR sequence is the ITR sequence of AAV2, comprising SEQ ID NOs: 8 and 9 and variants thereof. The AAV genome also generally includes packaging genes, such as the rep and/or cap genes, which encode packaging functions for AAV virus particles. The rep gene encodes one or more of Rep78, Rep68, Rep52 and Rep40 or variant proteins thereof. The cap gene encodes one or more capsid proteins such as VP1, VP2 and VP3 or variants thereof. These proteins make up the capsid of AAV virus particles. Variants of the capsid are described below.

The promoter is operably linked to each packaging gene. Specific examples of such promoters include the p5, p19 and p40 promoters (Laughlin et al., 1979, PNAS, 76:5567-5571). For example, the p40 promoter is generally used to express the cap gene, while the p5 and p19 promoters are commonly used to express the rep gene.

As previously reviewed, the AAV genome used in the vectors of the invention can thus be the entire genome of the native AAV virus. For example, vectors containing the entire AAV genome can be used to produce AAV viruses in vitro . However, such vectors can, in principle, be administered to patients, but will be rare in practice. Preferably, the AAV genome is deivatized for administration to the patient. This derivatization is standard in the art and the present invention includes the use of known derivatives of the AAV genome, and derivatives that can be generated by applying techniques known in the art. Derivatization of the AAV genome and derivatization of the AAV capsid are reviewed by Coura and Nardi (Virology Journal, 2007, 4:99), Choi et al. and Wu et al., cited above.

Derivatives of the AAV genome are in vivo ( in in vivo ) includes a truncated or modified form of the AAV genome that enables expression of the Rep-1 transgene in the vector of the invention. Generally, the AAV genome contains minimal viral sequence, but can be significantly cut to maintain the functions described above. This is desirable for safety reasons to reduce the risk of recombination of the vector and wild type virus, and also to avoid inducing a cellular immune response by the presence of viral gene proteins in target cells.

In general, the derivative comprises one or more inverted terminal repeat sequences (ITRs), preferably more than one ITR, such as two or more ITRs. The one or more ITRs may be from AAV genomes with different serotypes, or may be chimeric or mutant ITRs. The preferred mutant ITR is an ITR with deletion of trs (terminal resolution site). This deletion allows for continuous replication of a single-stranded genome that includes both coding and complementary sequences, that is, a genome to generate a self-complementary AAV genome. This enables bypass of DNA replication in the target cell, thus accelerating the expression of the transgene.

The one or more ITRs preferably flank the polynucleotide encoding REP1 or a variant thereof at both ends. Inclusion of one or more ITRs, for example, after conversion of single-stranded vector DNA to double-stranded DNA by the action of a host cell DNA polymerase, in the nucleus of the host cell, a concatamer of the vector of the invention Preferred to aid formation. The formation of these episomal serial chains protects the vector structure over the life of the host cell, thereby in vivo ( in In vivo ), it is possible to continuously express the transgene.

In a preferred embodiment, the ITR element will be the only sequence that is maintained from the native AAV genome in the derivative. Thus, the derivative will preferably not include the rep and/or cap genes of the native genome and any other sequence of the native genome. This is preferred for the reasons described above, and also reduces the possibility of vector fusion into the host cell genome. In addition, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (eg regulatory elements) in the vector in addition to the transgene.

For the AAV2 genome, part of the to of SEQ ID NO: 1, and can therefore be removed from the derivatives of the present invention: a single inverted terminal repeat (ITR) sequences, replication (rep) and capsid (cap) genes (NB: the wild-type AAV genome, Rep gene in human should not be confused with REP1, a human gene that affects pancreatic atrophy). However, in vitro (in In some embodiments, including in vitro ) embodiments, the derivative may additionally comprise one or more rep and/or cap genes or other viral sequences of the AAV genome. The native AAV virus is highly integrated at a specific site on human chromosome 19, and the frequency of random integration is negligible, so maintaining the ability to integrate in the vector can be tolerated in a therapeutic setting. have.

When the derivative genome comprises a gene encoding a capsid protein, ie VP1, VP2 and/or VP3, the derivative may be a chimeric derivative of one or more native AAV viruses, a shuffled derivative or a capsid-modified derivative. In particular, the present invention encompasses providing pseudo-typing, i.e., providing capsid protein sequences from different serotypes, lineages, clones, or isolates of AAV in the same vector.

Chimeric derivatives, shuffled derivatives, or capsid-modified derivatives are generally selected to provide one or more desired functions for the viral vector. Thus, these derivatives have increased efficiency of gene delivery, reduced immunogenicity (humoral or cellular) compared to AAV viral vectors comprising native AAV genomes, such as the genome of AAV2, altered range of migration activity of specific cell types and And/or improved targeting. The increased efficiency of gene delivery has improved receptor or co-receptor binding at the cell surface, improved internalisation, improved trafficking into cells and nuclei, and improved uncoating of viral particles. And improved conversion to the double-stranded form of the single-stranded genome. The increased efficiency is also related to the altered range of targeting or targeting of a particular cell population, so that the dose of the vector is not diluted by administration to tissues that are not needed.

Chimeric capsid proteins include capsid proteins produced by recombination between two or more capsid coding sequences of a native AAV serotype. This is, for example, that the non-infectious capsid sequence of one serotype is cotransfected with the capsid sequence of another serotype, and directed selection is used to select the capsid sequence with the desired properties. This can be done by a marker rescue approach. Capsid sequences of different serotypes can be altered by homologous recombination in cells to produce new chimeric capsid proteins.

Chimeric capsid proteins also genetically engineer capsid protein sequences to move specific capsid protein domains, surface loops or specific amino acid residues between two or more capsid proteins, e.g., between two or more different serotype capsid proteins. And capsid proteins produced by engineering.

Shuffled capsid proteins or chimeric capsid proteins can also be generated by DNA shuffling or error-prone PCR. The hybrid AAV capsid gene itself, which may randomly fragment the sequence of the gene encoding the related AAV gene, e.g., a capsid protein of several different serotypes, may then also cause crossover in the region of sequence homology. -Can also be produced by reassembling fragments in a priming polymerase reaction. A library of hybrid AAV genes produced in this way by shuffling the capsid genes of several serotypes can be selected to identify viral clones with the desired function. Similarly, error-prone PCR can be used to construct a diverse library of variants that can be selected for desired properties by randomly mutating AAV capsid genes.

The sequence of the capsid gene can also be genetically modified to introduce specific deletions, substitutions or insertions to the native wild type sequence. In particular, the capsid gene can be modified in an open reading frame of the capsid coding sequence or by insertion of a sequence of proteins or peptides not related to the N- and/or C-terminus of the capsid coding sequence.

Unrelated proteins or peptides may advantageously serve as ligands for a particular cell type, thereby conferring enhanced binding to target cells or enhancing the targeting specificity of the vector for a particular cell population. Examples may include the use of RGD peptides to block absorption in the retinal pigment epithelium and thereby enhance transduction of peripheral retinal tissue (Cronin et al., 2008 ARVO Abstract: D1048). The unrelated protein may also be a part of the production process that aids in the purification of viral particles, ie epitopes or affinity tags. The insertion site is generally selected so as not to interfere with other functions of the viral particle, such as internalization and migration of the viral particle. Those skilled in the art can identify appropriate sites for insertion based on common general knowledge. Certain sites are disclosed in Choi et al., cited above.

The invention also includes providing sequences of the AAV genome in a different order and configuration than those of the native AAV genome. The present invention also includes substituting one or more AAV sequences or genes with another viral sequence or a chimeric gene consisting of sequences from two or more viruses. These chimeric genes can consist of sequences from two or more related viral proteins of different viral species.

The vector of the present invention takes the form of a polynucleotide sequence comprising a sequence encoding an AAV genome or a derivative thereof and REP1 or a derivative thereof.

For the avoidance of doubt, the present invention also provides AAV virus particles comprising the vectors of the present invention. The AAV particles of the present invention include a transcapsidated form in which an AAV genome or derivative having one serotype ITR is packaged in different serotype capsids. The AAV particles of the present invention also include a mosaic form in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes a viral envelope. AAV particles also include chemically modified forms bearing ligands adsorbed to the capsid surface. For example, such ligands can include antibodies targeting specific cell surface receptors.

The invention also provides a host cell comprising the vector or AAV virus particle of the invention.

REP1

Vectors of the invention also include polynucleotide sequences encoding REP1 polypeptides or variants thereof. The human cDNA sequence for REP1 (or Rab escort protein-1, also known as Rab protein geranylgeranyltransferase component A) is shown in SEQ ID NO: 2 and sequenced The protein labeled No. 3 is encoded. Another cDNA sequence for Rep1 is represented by SEQ ID NO: 4.

The REP1 polypeptide or a variant thereof is a polypeptide that helps the prenylation of Rab GTPase protein. The ability of a REP1 polypeptide or variant thereof to help prenylation of a Rab GTP hydrolase protein can routinely be determined by one of skill in the art. The polynucleotide sequence encoding the variant of REP1 is a sequence encoding a protein that helps the prenylation activity of Rab-1 GTP hydrolase. Preferably, the sequence encodes a protein that helps provide similar or higher prenylation activity for Rab-1 GTP hydrolase compared to the polypeptide of SEQ ID NO: 3.

More preferably, the polynucleotide sequence encodes SEQ ID NO: 3 or a variant thereof, and is a variant of the polynucleotide sequence of SEQ ID NO: 2. Variants of SEQ ID NOs: 2 or 3 may include truncations, mutants or homologues thereof that encode functional REP polypeptides, and transcriptional variants thereof.

Homologs mentioned herein generally have at least 70% homology to the relevant region of SEQ ID NO: 2 or 3. Certain homologues are REP2 polypeptides that are 75% homologous to REP1, and can functionally compensate for REP1 deficiency.

Homology can be measured using known methods. For example, the UWGCG package provides a BESTFIT program that can be used to calculate homology (eg used in its default settings) (Devereux et al. (1984) Nucleic Acids Research 12, 387-395). See, eg, Altschul S.F. (1993) J Mol Evol 36:290-300; As described in Altschul, S. F. et al. (1990) J Mol Biol 215:403-10, the PILEUP and BLAST algorithms can be used to calculate homology or line up sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

In a preferred embodiment, the variant sequence is a sequence of 20 or more, preferably 30 or more, for example 40, 60, 100, 200, 300, 400 or more contiguous amino acids, or the entire sequence of the variant It is possible to encode a polypeptide that is at least 55%, 65%, 70%, 75%, 80%, 85%, 90% and more preferably at least 95%, 97% or 99% homologous to the relevant region of number 3. The relevant region will be a region that provides the functional activity of REP1 that helps the prenylation activity for Rab-1 GTP hydrolase.

Alternatively, and preferably, the variant sequence is full-lenth SEQ ID NO: 3 and 70%, 75%, 80%, 85%, 90% or more and more preferably 95% over the entire sequence, Polypeptides having 97% or 99% or more homology can be encoded. Generally, the variant sequence differs from the relevant region of SEQ ID NO: 3 by 2, 5, 10, 20, 40, 50 or 60 or more or less mutations, each of which may be a substitution, insertion or deletion.

The REP-1 polypeptide variant may have percent identity with a specific region of SEQ ID NO: 3, which is identical to a certain percentage homology value over the length of the aforementioned sequence (i.e., at least 70%, 80% or 90% and more Preferably 95%, 97% or 99% or more).

Variants of SEQ ID NO: 3 also include cleavage. The truncation can be used as long as the variant can prenylate the Rab-1 GTP hydrolase substrate polypeptide. Cleavage will generally be made to remove sequences that are non-essential to the prenylation activity and/or conformation of the folded protein, particularly the folding of the active site. Proper cleavage can be routinely identified by systematic truncation of sequences of varying lengths at the N- or C-terminus. The preferred cleavage is N-terminal and can remove all other sequences except the catalytic domain.

Variants of SEQ ID NO: 3 may also include one or more, for example, 2, 3, 4, 5 to 10, 10 to 20, 20 to 40 or more amino acid insertions, substitutions or deletions relative to a specific region of SEQ ID NO: 3. Mutants. Deletion and insertion preferably takes place outside the catalytic domain as described below. Substitutions also generally occur in regions that are non-essential to protease activity and/or do not affect the structure of the folded protein.

Substitutions preferably introduce one or more conservative changes, replacing amino acids with other chemicals of similar chemical structure, similar chemical properties or similar side-chain volumes. The introduced amino acids can have polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge similar to the amino acids they replace. Alternatively, conservative exchanges can introduce other amino acids that are aromatic or aliphatic, instead of the aromatic or aliphatic amino acids that already exist. Conservative amino acid exchange is well known in the art and can be selected according to the properties of the 20 major amino acids defined in Table A below.

Likewise, preferred variants of the polynucleotide sequence of SEQ ID NO: 2 are at least 70%, 75%, 80%, 85%, 90% or more and more preferably 95%, 97% or 99% or more homologous to the relevant region of SEQ ID NO:2 Phosphorus polynucleotides. Preferably, the variant exhibits this level of homology with the full-length SEQ ID NO: 2 over its entire sequence.

Table A-Chemical properties of amino acids Ala Aliphatic, hydrophobic, neutral Met Hydrophobic, neutral Cys Polar, hydrophobic, neutral Asn Polar, hydrophilic, neutral Asp Polarity, hydrophilicity, (-) charge Pro Hydrophobic, neutral Glu Polarity, hydrophilicity, (-) charge Gln Polar, hydrophilic, neutral Phe Aromatic, hydrophobic, neutral Arg Polarity, hydrophilicity, (+) charge Gly Aliphatic, neutral Ser Polar, hydrophilic, neutral His Aromatic, polar, hydrophilic, (+) charge Thr Polar, hydrophilic, neutral Ile Aliphatic, hydrophobic, neutral Val Aliphatic, hydrophobic, neutral Lys Polarity, hydrophilicity, (+) charge Trp Aromatic, hydrophobic, neutral Leu Aliphatic, hydrophobic, neutral Tyr Aromatic, polar, hydrophobic

Promoter and regulatory sequences

The vectors of the present invention also include elements that enable the expression of the REP1 transgene in vitro or in vivo. Thus, vectors generally include a promoter sequence operably linked to a polynucleotide sequence encoding Rep-1 or a variant thereof.

Any suitable promoter can be used. The promoter sequence can be constitutively active, ie operable in any host cell environment, or alternatively, can be activated only in a particular host cell environment, targeting the transgene in a particular cell type. Enables expression. The promoter may exhibit inducible expression in response to the presence of another factor, such as a factor present in the host cell. When the vector is administered for treatment, the promoter must operate in a retinal cell environment.

In some embodiments, it is preferred that the promoter exhibits retinal-cell specific expression so that the transgene is expressed only in the retinal cell population. Thus, expression from that promoter can be retinal-cell specific, for example, only confined to cells of the retinal optic nerve and retinal pigment epithelium.

Preferred promoters for the Rep-1 transgene include the chicken beta-actin (CBA) promoter, optionally combined with a cytomegalovirus (CME) enhancer element. Particularly preferred promoters are hybrid CBA/CAG promoters, such as those used in the rAVE expression cassette (GeneDetect.com). Also, a preferred promoter is represented by SEQ ID NO: 6. Examples of promoters based on human sequences that induce retinal specific gene expression include rhodospin kinase for rods and abstracts (Allocca et al., 2007, J Virol 81:11372-80), PR2 for abstracts alone. 1 (Mancuso et al., 2009, Nature) and/or RPE65 for retinal pigment epithelium (Bainbridge et al., 2008, N Eng J Med).

The vectors of the invention can also include one or more additional regulatory sequences that can act before or after transcription. The regulatory sequence can be part of the native REP1 locus or can be a heterologous regulatory sequence. The vectors of the present invention may contain a portion of 5'UTR or 3'UTR from a native REP1 transcript. For example, the polynucleotide of SEQ ID NO: 4 comprises a portion of the 5'UTR sequence from the native REP1 transcript.

Regulatory sequences are sequences that promote the expression of transgenes, that is, sequences that increase expression of transcripts, enhance nuclear export of mRNA, or enhance their stability. Such regulatory sequences include, for example, enhancer elements, postregulatory elements and polyadenylation sites. A preferred polyadenylation site is the Bovine Growth Hormone poly-A signal, which may be as shown in SEQ ID NO: 7. In the vector context of the invention, this regulatory sequence will be cis-functional. However, the present invention also encompasses the use of trans-acting regulatory sequences located in additional genetic constructs.

Preferred post-regulatory elements for use in the vectors of the invention are woodchuck hepatitis post-regulatory elements (WPRE) or variants thereof. The sequence of WPRE is provided in SEQ ID NO: 5. The present invention includes the use of variant sequences of WPRE that increase expression of the REP1 transgene compared to vectors without WPRE. Preferably, the variant sequence has at least 70% homology with SEQ ID NO: 5 over its entire sequence, more preferably at least 75%, 80%, 85%, 90% homology, more preferably over its entire sequence It shows at least 95%, 97%, or 99% homology with SEQ ID NO:5.

Another regulatory sequence that can be used in the vectors of the present invention is the scaffold-attachment region (SAR). Additional regulatory sequences can be selected by those skilled in the art based on common and general knowledge.

Preparation of vector

Vectors of the present invention can be prepared by standard means known in the art to provide vectors for gene therapy. Thus, established public domain transfection, packaging and purification methods can be used to prepare appropriate vectors.

As previously reviewed, the vectors of the present invention may include the entire genome of the native AAV virus as well as the polynucleotide encoding REP1 or a variant thereof. However, derivatives that usually have a derivatised genome, such as one or more inverted terminal repeat sequences (ITRs), but which may lack the AAV gene, such as rep or cap , will be used.

In this embodiment, to provide assembly of the derivatized genome into AAV virus particles, additional genetic constructs that provide AAV and/or helper virus function will be provided to the host cell along with the derivatized genome. will be. Such additional constructs generally include genes encoding structural AAV capsid proteins, i.e., cap , VP1, VP2, VP3, and genes encoding other functions required for AAV life cycle, such as rep . The choice of structural capsid protein provided to the additional construct will determine the serotype of the packaged viral vector.

A particularly preferred packaged viral vector for use in the present invention comprises a derivatized genome of AAV2 along with an AAV5 or AAV8 capsid protein. This packaging viral vector generally comprises one or more AAV2 ITRs, or variants thereof, optionally represented by SEQ ID NOs: 8 and/or 9.

As mentioned above, AAV viruses are not capable of replication, and thus helper virus function, preferably adenovirus helper function, will also be provided to one or more additional structures to enable AAV replication.

All of the additional structures described above may be provided as plasmids or other episomal elements of the host cell, or alternatively, one or more structures may be incorporated into the genome of the host cell.

In this aspect, the present invention provides a method of making the vector of the present invention. The method provides a host cell with a vector comprising a polypeptide sequence encoding an adeno-associated virus (AAV) genome or a derivative thereof and a REP1 or variant thereof, a means for replicating the vector and assembling it into AAV virus particles It includes the steps. Preferably, the method comprises providing a vector comprising a polynucleotide sequence encoding a derivative of the AAV genome and a REP1 or variant thereof together with one or more additional genetic constructs encoding AAV and/or helper virus function. do. In general, derivatives of the AAV genome include one or more ITRs. Optionally, the method also includes the step of purifying the assembled viral particles. In addition, the method may include formulating viral particles for therapeutic use.

Treatment method and medicine use

As previously reviewed, the inventors have surprisingly shown that the vectors of the present invention can be used to address cellular dysfunction inherent in pancreatic atrophy. In particular, they have shown that the use of vectors can correct frenylation defects associated with pancreatic atrophy. It provides a means to treat, arrest, palliated or prevent disease degeneration.

The present invention thus treats pancreatic atrophy in a patient in need of treatment or prevention of pancreatic atrophy, comprising administering to the patient a therapeutically effective amount of the vector of the invention by direct retinal injection, subretinal injection or intravitreal injection. Or provide a way to prevent it. Thus, pancreatic atrophy is thereby treated or prevented in the patient.

In a related aspect, the present invention provides the use of the vector of the present invention in a method of treating or preventing pancreatic atrophy by administering said vector to a patient by direct retinal injection, subretinal injection or intravitreal injection. In addition, the present invention provides the use of the vector of the invention in the manufacture of a drug that treats or prevents pancreatic atrophy by direct retinal injection, subretinal injection or intravitreal injection.

In all these embodiments, the vectors of the invention can be administered to prevent onset of one or more symptoms of pancreatic atrophy. The patient may be asymptomatic. Subject may have predisposition to pancreatic atrophy. The method or use may include determining whether the subject is at risk of developing pancreatic atrophy or has pancreatic atrophy. A prophylactically effective amount of vector is administered to these subjects. A prophylactically effective amount is an amount that prevents the onset of one or more symptoms of the disease.

Alternatively, the vector can be administered when symptoms of the disease appear on the subject, ie to treat the existing symptoms of the disease. A therapeutically effective amount of the antagonist is administered to these subjects. A therapeutically effective amount is an amount effective to improve one or more symptoms of a disease. Generally, this amount increases the level of prenylation of Rab GTP hydrolase in the eye. This can be confirmed as described below. This amount can stop, slow or reverse the loss of peripheral vision associated with pancreatic atrophy. This amount can also stop, slow or reverse the onset of night blindness.

The subject can be male or female. Because panchoroidal atrophy is an X-chromosome-related disease, men have more severe symptoms, but women also have symptoms and sometimes severe phenotypes. The subject is preferably identified as at risk or at risk of the disease. The retina may first have a characteristic appearance that thins the choroid and partially proceeds with exposure of the sclera below it. There may be a loss of amplitude of the retinal potential at the peripherally. In many cases, there may be a family history of pancreatic atrophy. Generally, but not always, mutations can be identified in the REP1 gene located on the X-chromosome.

Administration of the vector is usually by direct retinal injection or subretinal injection. This includes direct delivery to the retinal pigment epithelial cells and neurosensory retina, such as epithelial cells or photoreceptor cells. This delivery is usually direct or subretinal to the degenerative retina of a patient with pancreatic atrophy. Vectors can be transduced into the target cells described above without entering other cell populations. Intravitreal injections can also be used to deliver the vectors of the invention. Delivery may not be subretinal or may be by subretinal injection. Delivery may not transvitreal.

Dosages of the vectors of the present invention may vary in various parameters, particularly age, weight and condition of the patient to be treated; Route of administration; And the required regimen (regimen). In addition, the doctor can determine the route and dose required for a particular patient.

Depending on the amount of retinal tissue remaining to be transduced, a typical single dose is 10 ¹⁰ to 10 ¹² genomic particles (gp). Genome particles are defined herein as AAV capsids containing single-stranded DNA molecules that can be quantified by sequence-specific methods (eg, real-time PCR). This dose can be given as a single dose, but can be repeated for the opposite eye or for some reason (eg, surgical complications) if the vector fails to target the correct region of the retina. The treatment is preferably a one-time permanent treatment for each eye, but repeated injections, for example after several years and/or with other AAV serotypes, can be considered.

The present invention also includes the step of measuring ex vivo prenylation activity in retinal cells obtained from a patient after administering the AAV vector of the present invention by direct retinal injection, subretinal injection or intravitreal injection, in a patient. It provides a method of monitoring the treatment or prevention of pancreatic atrophy. This method allows to determine the effectiveness of treatment.

Pharmaceutical composition

Vectors of the invention can be formulated into pharmaceutical compositions. Such compositions may include, in addition to vectors, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. These substances should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material can be determined by one skilled in the art depending on the route of administration, ie direct retinal injection, subretinal injection, intravitreal injection.

The pharmaceutical composition is generally in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal oil or vegetable oil, mineral oil or synthetic oil. Physiological saline solutions, magnesium chloride, dextrose or other sugar solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, surfactants such as 0.001% of pluronic acid (PF68) can be used.

For injection into the affected area, the active ingredient will be in the form of an aqueous solution that is pyrogen-free and has adequate pH, isotonic and stability. A person skilled in the art can prepare an appropriate solution well using isotonic vehicles such as sodium chloride injection solution, Ringer injection solution, and lactated Ringer's Injection. If necessary, preservatives, stabilizers, buffers, antioxidants and/or other additives may be included.

For delayed release, the vector is a microcapsule or liposome carrier system formed from a biocompatible polymer, e.g., in a pharmaceutical composition formulated for slow release, according to methods known in the art. Can be included in

Example

This example describes a model for evaluating treatment strategies for pancreatic atrophy and correction of pancreatic atrophy phenotype. When a genetic construct consisting of a promoter, REP1 cDNA, and 3'regulatory element is packaged with a recombinant viral vector, it appears to be efficiently transduced into target cells in the denatured retina.

Example One

human REP1 cDNA of Cloning , And CBA - REP -One- WPRE Generation of expression cassettes, pAAV -CBA-REP-1-WPRE-bGHpA construction and AAV REP -1 packaging of viruses.

The cDNA of human REP1 was isolated from human cDNA library using PCR amplification and primers homologous to known REP1 sequences. The sequence of the isolated cDNA was determined and confirmed to be homologous to the known translated variant 1 of the REP1 mRNA sequence deposited with Genebank under accession number NM_000390. This cDNA has the sequence of SEQ ID NO: 4.

This cDNA was inserted into a pAAV cis plasmid called pAM. pAM is a plasmid with a high copy number originally derived from pBR322, but contains stabilized AAV-2 left and right inverted terminal repeats flanking the selected expression cassette. In the AAV-REP1 vector, a modified CBA/CAG promoter (chicken beta-actin with CMV enhancer) was used to drive expression of REP1 and modified WPRE sequences and bGH polyA was provided 3'to cDNA. . This plasmid was named pAAV2-CBA-hREP-1-WPRE-bGH, (pAAV-REP-1).

pAAV-REP-1 was used to generate recombinant AAV-Rep-1 using a triple transfection packaging and purification method in an established public domain. The method vector stock solution (stock) generated using almost the various genome titer (titer), the stock solution obtained after purification of the most commonly is 10 ¹² - 10 ¹³ gp / ml was (= gp genome particles - see above). This stock solution was then diluted for in vivo use as described below.

Example 2

human Pancreatic atrophy ( Chm ) From vectors in cells REP1 Manifestation of

Expression of REP1 from the AAV2.REP1 vector was evaluated in human pancreatic atrophy (Chm) fibroblasts. These fibroblasts were obtained from skin biopsies with an ethical agreement from patients with pancreatic atrophy. Expression of GFP from the control vector served as a control. In preparation for the study by human cells, the expression of the antibody after the subretinal injection of the AAV.REP1 vector into the mouse was also confirmed by Western blot, as the antibody Fvorre recognizes the human form of the REP1 protein but not the mouse form.

The results are shown in FIG. 1. REP1 was not detected by immunoblotting with anti-hREP1 antibody in nontransduced Chm fibroblasts (lane 2), while REP1 was normal (WT=human wild type) fibroblasts (lane 1) ). After transduction with the AAV2.REP1 vector, the level of hREP1 expressed by the AAV2.REP1 vector in Chm cells at the same dose of 40 μg lysate and 5 μg dilution was approximately 10 times that of wild-type cells (lane 1). It can be seen that it is higher (lanes 3-6). The absence of toxic effects on cell growth was also observed with the extent of this over-expression.

Example 3

Chm In cells by vector Prenylation Correction of defects

Because panchoroidal mice do not have the retinal degeneration phenotype in the same way as human patients, it is impossible to perform a direct assessment of retinal repair using a gene therapy approach. For this reason, correction of pancreatic atrophy phenotype was evaluated in human Chm cells in vitro.

The results are shown in FIG. 2. Transduction with AAV2.REP1 shows correction of the prenylation defects seen in Chm cells, AAV2. After treatment of 2 x 10 ⁵ cells with 1.5 x 10 ¹⁰ viral genomic particles of REP1, the prenylation activity was significantly higher than normal level. This confirms that the AAV2.REP1 vector expresses a functional REP1 protein in human cells affected by pancreatic atrophy.

More specifically, normal prenylation activity in wild-type (WT) fibroblasts yielded about 0.32 pmol of [3H]-GGPP; In pancreatic atrophy (Chm) fibroblasts, it decreases to 0.19 pmol. As expected, the prenylation activity did not change after transduction of AAV.GFP control vector into Chm fibroblasts. However, after transduction with the AAV2.REP1 vector, the prenylation activity increased significantly (n=4, p<0.01), yielding 0.42 pmol of [3H]-GGPP.

Example 4

In vivo of reporter gene from vector in mouse Targeted Manifestation

In order to confirm the ubiquitous activity of the CBA promoter and regulatory sequence in the AAV2 vector, a gene encoding REP1 is encoded with a green fluorescent protein (GFP) to make AAV2.CBA.GFP.WPRE.BGH (AAV2.GFP). Was replaced with a reporter gene. Human REP1 protein was easily identified by Western blot, but GFP was selected to evaluate expression in vivo because it was not readily detected by indirect immunohistochemistry in the retinal section.

The AAV2.GFP construct was injected into the subretinal space of the mouse and the expression of GFP was monitored microscopically. The results are shown in FIG. 3 confirming that the vector has the expected migration activity in both the retinal optic nerve and the retinal pigment epithelium. This confirms that the capsid sequence and regulatory elements result in high levels of gene expression in photoreceptors and retinal pigment epithelium.

Example 5

Toxicity studies

To determine possible toxic effects on retinal function at the highest dose, the dose of the AAV2.REP1 vector was injected into the subretinal space of wild-type mice (n=9). The inventors tested vector concentrations in log units (gps per ml 1 x 10 ¹¹ and 1 x 10 ¹² ml per ml) above the high and low concentrations (10 ¹⁰ and 10 ¹¹ gp per ml) suggested to be used in patients in mice. Did.

The results are shown in FIG. 4. No toxic effect was detected in retinal potential (ERG) for 6 months after subretinal injection with either the high dose (n=5) or low dose (n=4) of the AAV.REP1 vector. To modulate retinal surgery or the non-specific effects of the AAV2 vector, the opposite eye was injected subtiterly with the titer of AAV2.GFP very similarly.

There was a mild decrease in ERG amplitude at the highest dose of AAV2.GFP, reflecting a known minor toxic effect using the maximum dose of the vector expressing GFP with this potent promoter, and dose-related effects The sensitivity of this test to the detection of effects) is confirmed. Nevertheless, there was no detectable ERG reduction in the eyes treated with AAV2.REP1 at high or low doses, suggesting that REP1 over-expression in the retina was less toxic than GFP.

<110> ISIS INNOVATION LIMITED <120> METHOD <130> N.112542A LPC <150> GB 1103062.4 <151> 2011-02-22 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 4679 <212> DNA <213> adeno-associated virus 2 <400> 1 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120 gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtgaat tacgtcatag 180 ggttagggag gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat 240 gtggtcacgc tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga 300 ggtttgaacg cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg 360 accttgacga gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg 420 aatgggagtt gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga 480 ccgtggccga gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc 540 cggaggccct tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc 600 tcgtggaaac caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg 660 aaaaactgat tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg 720 tcacaaagac cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc 780 ccaattactt gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac 840 agtatttaag cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga 900 cgcacgtgtc gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc 960 cggtgatcag atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca 1020 aggggattac ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca 1080 atgcggcctc caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta 1140 tgagcctgac taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt 1200 ccagcaatcg gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt 1260 ccgtctttct gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg 1320 ggcctgcaac taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct 1380 acgggtgcgt aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg 1440 tgatctggtg ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc 1500 tcggaggaag caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga 1560 ctcccgtgat cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga 1620 ccttcgaaca ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc 1680 tggatcatga ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa 1740 aggatcacgt ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa 1800 gacccgcccc cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc 1860 agccatcgac gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat 1920 gttctcgtca cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga 1980 atcagaattc aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg 2040 tgtcagaatc tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc 2100 atcatatcat gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt 2160 tggatgactg catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat 2220 cttccagatt ggctcgagga cactctctct gaaggaataa gacagtggtg gaagctcaaa 2280 cctggcccac caccaccaaa gcccgcagag cggcataagg acgacagcag gggtcttgtg 2340 cttcctgggt acaagtacct cggacccttc aacggactcg acaagggaga gccggtcaac 2400 gaggcagacg ccgcggccct cgagcacgac aaagcctacg accggcagct cgacagcgga 2460 gacaacccgt acctcaagta caaccacgcc gacgcggagt ttcaggagcg ccttaaagaa 2520 gatacgtctt ttgggggcaa cctcggacga gcagtcttcc aggcgaaaaa gagggttctt 2580 gaacctctgg gcctggttga ggaacctgtt aagacggctc cgggaaaaaa gaggccggta 2640 gagcactctc ctgtggagcc agactcctcc tcgggaaccg gaaaggcggg ccagcagcct 2700 gcaagaaaaa gattgaattt tggtcagact ggagacgcag actcagtacc tgacccccag 2760 cctctcggac agccaccagc agccccctct ggtctgggaa ctaatacgat ggctacaggc 2820 agtggcgcac caatggcaga caataacgag ggcgccgacg gagtgggtaa ttcctcggga 2880 aattggcatt gcgattccac atggatgggc gacagagtca tcaccaccag cacccgaacc 2940 tgggccctgc ccacctacaa caaccacctc tacaaacaaa tttccagcca atcaggagcc 3000 tcgaacgaca atcactactt tggctacagc accccttggg ggtattttga cttcaacaga 3060 ttccactgcc acttttcacc acgtgactgg caaagactca tcaacaacaa ctggggattc 3120 cgacccaaga gactcaactt caagctcttt aacattcaag tcaaagaggt cacgcagaat 3180 gacggtacga cgacgattgc caataacctt accagcacgg ttcaggtgtt tactgactcg 3240 gagtaccagc tcccgtacgt cctcggctcg gcgcatcaag gatgcctccc gccgttccca 3300 gcagacgtct tcatggtgcc acagtatgga tacctcaccc tgaacaacgg gagtcaggca 3360 gtaggacgct cttcatttta ctgcctggag tactttcctt ctcagatgct gcgtaccgga 3420 aacaacttta ccttcagcta cacttttgag gacgttcctt tccacagcag ctacgctcac 3480 agccagagtc tggaccgtct catgaatcct ctcatcgacc agtacctgta ttacttgagc 3540 agaacaaaca ctccaagtgg aaccaccacg cagtcaaggc ttcagttttc tcaggccgga 3600 gcgagtgaca ttcgggacca gtctaggaac tggcttcctg gaccctgtta ccgccagcag 3660 cgagtatcaa agacatctgc ggataacaac aacagtgaat actcgtggac tggagctacc 3720 aagtaccacc tcaatggcag agactctctg gtgaatccgg gcccggccat ggcaagccac 3780 aaggacgatg aagaaaagtt ttttcctcag agcggggttc tcatctttgg gaagcaaggc 3840 tcagagaaaa caaatgtgga cattgaaaag gtcatgatta cagacgaaga ggaaatcagg 3900 acaaccaatc ccgtggctac ggagcagtat ggttctgtat ctaccaacct ccagagaggc 3960 aacagacaag cagctaccgc agatgtcaac acacaaggcg ttcttccagg catggtctgg 4020 caggacagag atgtgtacct tcaggggccc atctgggcaa agattccaca cacggacgga 4080 cattttcacc cctctcccct catgggtgga ttcggactta aacaccctcc tccacagatt 4140 ctcatcaaga acaccccggt acctgcgaat ccttcgacca ccttcagtgc ggcaaagttt 4200 gcttccttca tcacacagta ctccacggga caggtcagcg tggagatcga gtgggagctg 4260 cagaaggaaa acagcaaacg ctggaatccc gaaattcagt acacttccaa ctacaacaag 4320 tctgttaatg tggactttac tgtggacact aatggcgtgt attcagagcc tcgccccatt 4380 ggcaccagat acctgactcg taatctgtaa ttgcttgtta atcaataaac cgtttaattc 4440 gtttcagttg aactttggtc tctgcgtatt tctttcttat ctagtttcca tggctacgta 4500 gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc 4560 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc 4620 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaa 4679 <210> 2 <211> 1962 <212> DNA <213> Homo sapiens <400> 2 atggcggata ctctcccttc ggagtttgat gtgatcgtaa tagggacggg tttgcctgaa 60 tccatcattg cagctgcatg ttcaagaagt ggccggagag ttctgcatgt tgattcaaga 120 agctactatg gaggaaactg ggccagtttt agcttttcag gactattgtc ctggctaaag 180 gaataccagg aaaacagtga cattgtaagt gacagtccag tgtggcaaga ccagatcctt 240 gaaaatgaag aagccattgc tcttagcagg aaggacaaaa ctattcaaca tgtggaagta 300 ttttgttatg ccagtcagga tttgcatgaa gatgtcgaag aagctggtgc actgcagaaa 360 aatcatgctc ttgtgacatc tgcaaactcc acagaagctg cagattctgc cttcctgcct 420 acggaggatg agtcattaag cactatgagc tgtgaaatgc tcacagaaca aactccaagc 480 agcgatccag agaatgcgct agaagtaaat ggtgctgaag tgacagggga aaaagaaaac 540 cattgtgatg ataaaacttg tgtgccatca acttcagcag aagacatgag tgaaaatgtg 600 cctatagcag aagataccac agagcaacca aagaaaaaca gaattactta ctcacaaatt 660 attaaagaag gcaggagatt taatattgat ttagtatcaa agctgctgta ttctcgagga 720 ttactaattg atcttctaat caaatctaat gttagtcgat atgcagagtt taaaaatatt 780 accaggattc ttgcatttcg agaaggacga gtggaacagg ttccgtgttc cagagcagat 840 gtctttaata gcaaacaact tactatggta gaaaagcgaa tgctaatgaa atttcttaca 900 ttttgtatgg aatatgagaa atatcctgat gaatataaag gatatgaaga gatcacattt 960 tatgaatatt taaagactca aaaattaacc cccaacctcc aatatattgt catgcattca 1020 attgcaatga catcagagac agccagcagc accatagatg gtctcaaagc taccaaaaac 1080 tttcttcact gtcttgggcg gtatggcaac actccatttt tgtttccttt atatggccaa 1140 ggagaactcc cccagtgttt ctgcaggatg tgtgctgtgt ttggtggaat ttattgtctt 1200 cgccattcag tacagtgcct tgtagtggac aaagaatcca gaaaatgtaa agcaattata 1260 gatcagtttg gtcagagaat aatctctgag catttcctcg tggaggacag ttactttcct 1320 gagaacatgt gctcacgtgt gcaatacagg cagatctcca gggcagtgct gattacagat 1380 agatctgtcc taaaaacaga ttcagatcaa cagatttcca ttttgacagt gccagcagag 1440 gaaccaggaa cttttgctgt tcgggtcatt gagttatgtt cttcaacgat gacatgcatg 1500 aaaggcacct atttggttca tttgacttgc acatcttcta aaacagcaag agaagattta 1560 gaatcagttg tgcagaaatt gtttgttcca tatactgaaa tggagataga aaatgaacaa 1620 gtagaaaagc caagaattct gtgggctctt tacttcaata tgagagattc gtcagacatc 1680 agcaggagct gttataatga tttaccatcc aacgtttatg tctgctctgg cccagattgt 1740 ggtttaggaa atgataatgc agtcaaacag gctgaaacac ttttccagga aatctgcccc 1800 aatgaagatt tctgtccccc tccaccaaat cctgaagaca ttatccttga tggagacagt 1860 ttacagccag aggcttcaga atccagtgcc ataccagagg ctaactcgga gactttcaag 1920 gaaagcacaa accttggaaa cctagaggag tcctctgaat aa 1962 <210> 3 <211> 653 <212> PRT <213> Homo sapiens <400> 3 Met Ala Asp Thr Leu Pro Ser Glu Phe Asp Val Ile Val Ile Gly Thr 1 5 10 15 Gly Leu Pro Glu Ser Ile Ile Ala Ala Ala Cys Ser Arg Ser Gly Arg 20 25 30 Arg Val Leu His Val Asp Ser Arg Ser Tyr Tyr Gly Gly Asn Trp Ala 35 40 45 Ser Phe Ser Phe Ser Gly Leu Leu Ser Trp Leu Lys Glu Tyr Gln Glu 50 55 60 Asn Ser Asp Ile Val Ser Asp Ser Pro Val Trp Gln Asp Gln Ile Leu 65 70 75 80 Glu Asn Glu Glu Ala Ile Ala Leu Ser Arg Lys Asp Lys Thr Ile Gln 85 90 95 His Val Glu Val Phe Cys Tyr Ala Ser Gln Asp Leu His Glu Asp Val 100 105 110 Glu Glu Ala Gly Ala Leu Gln Lys Asn His Ala Leu Val Thr Ser Ala 115 120 125 Asn Ser Thr Glu Ala Ala Asp Ser Ala Phe Leu Pro Thr Glu Asp Glu 130 135 140 Ser Leu Ser Thr Met Ser Cys Glu Met Leu Thr Glu Gln Thr Pro Ser 145 150 155 160 Ser Asp Pro Glu Asn Ala Leu Glu Val Asn Gly Ala Glu Val Thr Gly 165 170 175 Glu Lys Glu Asn His Cys Asp Asp Lys Thr Cys Val Pro Ser Thr Ser 180 185 190 Ala Glu Asp Met Ser Glu Asn Val Pro Ile Ala Glu Asp Thr Thr Glu 195 200 205 Gln Pro Lys Lys Asn Arg Ile Thr Tyr Ser Gln Ile Ile Lys Glu Gly 210 215 220 Arg Arg Phe Asn Ile Asp Leu Val Ser Lys Leu Leu Tyr Ser Arg Gly 225 230 235 240 Leu Leu Ile Asp Leu Leu Ile Lys Ser Asn Val Ser Arg Tyr Ala Glu 245 250 255 Phe Lys Asn Ile Thr Arg Ile Leu Ala Phe Arg Glu Gly Arg Val Glu 260 265 270 Gln Val Pro Cys Ser Arg Ala Asp Val Phe Asn Ser Lys Gln Leu Thr 275 280 285 Met Val Glu Lys Arg Met Leu Met Lys Phe Leu Thr Phe Cys Met Glu 290 295 300 Tyr Glu Lys Tyr Pro Asp Glu Tyr Lys Gly Tyr Glu Glu Ile Thr Phe 305 310 315 320 Tyr Glu Tyr Leu Lys Thr Gln Lys Leu Thr Pro Asn Leu Gln Tyr Ile 325 330 335 Val Met His Ser Ile Ala Met Thr Ser Glu Thr Ala Ser Ser Thr Ile 340 345 350 Asp Gly Leu Lys Ala Thr Lys Asn Phe Leu His Cys Leu Gly Arg Tyr 355 360 365 Gly Asn Thr Pro Phe Leu Phe Pro Leu Tyr Gly Gln Gly Glu Leu Pro 370 375 380 Gln Cys Phe Cys Arg Met Cys Ala Val Phe Gly Gly Ile Tyr Cys Leu 385 390 395 400 Arg His Ser Val Gln Cys Leu Val Val Asp Lys Glu Ser Arg Lys Cys 405 410 415 Lys Ala Ile Ile Asp Gln Phe Gly Gln Arg Ile Ile Ser Glu His Phe 420 425 430 Leu Val Glu Asp Ser Tyr Phe Pro Glu Asn Met Cys Ser Arg Val Gln 435 440 445 Tyr Arg Gln Ile Ser Arg Ala Val Leu Ile Thr Asp Arg Ser Val Leu 450 455 460 Lys Thr Asp Ser Asp Gln Gln Ile Ser Ile Leu Thr Val Pro Ala Glu 465 470 475 480 Glu Pro Gly Thr Phe Ala Val Arg Val Ile Glu Leu Cys Ser Ser Thr 485 490 495 Met Thr Cys Met Lys Gly Thr Tyr Leu Val His Leu Thr Cys Thr Ser 500 505 510 Ser Lys Thr Ala Arg Glu Asp Leu Glu Ser Val Val Gln Lys Leu Phe 515 520 525 Val Pro Tyr Thr Glu Met Glu Ile Glu Asn Glu Gln Val Glu Lys Pro 530 535 540 Arg Ile Leu Trp Ala Leu Tyr Phe Asn Met Arg Asp Ser Ser Asp Ile 545 550 555 560 Ser Arg Ser Cys Tyr Asn Asp Leu Pro Ser Asn Val Tyr Val Cys Ser 565 570 575 Gly Pro Asp Cys Gly Leu Gly Asn Asp Asn Ala Val Lys Gln Ala Glu 580 585 590 Thr Leu Phe Gln Glu Ile Cys Pro Asn Glu Asp Phe Cys Pro Pro Pro 595 600 605 Pro Asn Pro Glu Asp Ile Ile Leu Asp Gly Asp Ser Leu Gln Pro Glu 610 615 620 Ala Ser Glu Ser Ser Ala Ile Pro Glu Ala Asn Ser Glu Thr Phe Lys 625 630 635 640 Glu Ser Thr Asn Leu Gly Asn Leu Glu Glu Ser Ser Glu 645 650 <210> 4 <211> 1992 <212> DNA <213> Homo sapiens <400> 4 gatatcgaat tcctgcagcc cggcggcacc atggcggata ctctcccttc ggagtttgat 60 gtgatcgtaa tagggacggg tttgcctgaa tccatcattg cagctgcatg ttcaagaagt 120 ggccggagag ttctgcatgt tgattcaaga agctactatg gaggaaactg ggccagtttt 180 agcttttcag gactattgtc ctggctaaag gaataccagg aaaacagtga cattgtaagt 240 gacagtccag tgtggcaaga ccagatcctt gaaaatgaag aagccattgc tcttagcagg 300 aaggacaaaa ctattcaaca tgtggaagta ttttgttatg ccagtcagga tttgcatgaa 360 gatgtcgaag aagctggtgc actgcagaaa aatcatgctc ttgtgacatc tgcaaactcc 420 acagaagctg cagattctgc cttcctgcct acggaggatg agtcattaag cactatgagc 480 tgtgaaatgc tcacagaaca aactccaagc agcgatccag agaatgcgct agaagtaaat 540 ggtgctgaag tgacagggga aaaagaaaac cattgtgatg ataaaacttg tgtgccatca 600 acttcagcag aagacatgag tgaaaatgtg cctatagcag aagataccac agagcaacca 660 aagaaaaaca gaattactta ctcacaaatt attaaagaag gcaggagatt taatattgat 720 ttagtatcaa agctgctgta ttctcgagga ttactaattg atcttctaat caaatctaat 780 gttagtcgat atgcagagtt taaaaatatt accaggattc ttgcatttcg agaaggacga 840 gtggaacagg ttccgtgttc cagagcagat gtctttaata gcaaacaact tactatggta 900 gaaaagcgaa tgctaatgaa atttcttaca ttttgtatgg aatatgagaa atatcctgat 960 gaatataaag gatatgaaga gatcacattt tatgaatatt taaagactca aaaattaacc 1020 cccaacctcc aatatattgt catgcattca attgcaatga catcagagac agccagcagc 1080 accatagatg gtctcaaagc taccaaaaac tttcttcact gtcttgggcg gtatggcaac 1140 actccatttt tgtttccttt atatggccaa ggagaactcc cccagtgttt ctgcaggatg 1200 tgtgctgtgt ttggtggaat ttattgtctt cgccattcag tacagtgcct tgtagtggac 1260 aaagaatcca gaaaatgtaa agcaattata gatcagtttg gtcagagaat aatctctgag 1320 catttcctcg tggaggacag ttactttcct gagaacatgt gctcacgtgt gcaatacagg 1380 cagatctcca gggcagtgct gattacagat agatctgtcc taaaaacaga ttcagatcaa 1440 cagatttcca ttttgacagt gccagcagag gaaccaggaa cttttgctgt tcgggtcatt 1500 gagttatgtt cttcaacgat gacatgcatg aaaggcacct atttggttca tttgacttgc 1560 acatcttcta aaacagcaag agaagattta gaatcagttg tgcagaaatt gtttgttcca 1620 tatactgaaa tggagataga aaatgaacaa gtagaaaagc caagaattct gtgggctctt 1680 tacttcaata tgagagattc gtcagacatc agcaggagct gttataatga tttaccatcc 1740 aacgtttatg tctgctctgg cccagattgt ggtttaggaa atgataatgc agtcaaacag 1800 gctgaaacac ttttccagga aatctgcccc aatgaagatt tctgtccccc tccaccaaat 1860 cctgaagaca ttatccttga tggagacagt ttacagccag aggcttcaga atccagtgcc 1920 ataccagagg ctaactcgga gactttcaag gaaagcacaa accttggaaa cctagaggag 1980 tcctctgaat aa 1992 <210> 5 <211> 588 <212> DNA <213> Woodchuck hepatitis B virus <400> 5 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc 60 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta 120 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt 180 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg 240 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta 300 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt 360 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg 420 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca 480 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc 540 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgc 588 <210> 6 <211> 934 <212> DNA <213> Gallus gallus <400> 6 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 60 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 120 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 180 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 240 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 300 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 360 gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 420 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 480 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg 540 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 600 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 660 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct 720 ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc 780 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag 840 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg 900 ttattgtgct gtctcatcat tttggcaaag aatt 934 <210> 7 <211> 270 <212> DNA <213> Bos primigenius <400> 7 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 60 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 120 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 180 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 240 ggcttctgag gcggaaagaa ccagctgggg 270 <210> 8 <211> 144 <212> DNA <213> adeno-associated virus 2 <400> 8 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 aggggttcct tgtagttaat gatt 144 <210> 9 <211> 145 <212> DNA <213> adeno-associated virus 2 <400> 9 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 60 cccgggcggc ctcagtgagc gagcgagcgc gcagagcttt ttgcaaaagc ctaggcctcc 120 aaaaaagcct cctcactact tctgg 145

Claims (34)

A polynucleotide sequence encoding a REP1 (Rab escort protein 1) polypeptide having at least 97% identity with SEQ ID NO: 3, wherein the polynucleotide is operably linked to a chicken beta actin (CBA) promoter Adeno-associated virus (AAV) vectors. The vector according to claim 1, wherein the AAV is a naturally-derived serotype or isolate or clade of AAV. The vector of claim 2, wherein the serotype is AAV serotype 2 (AAV2). The method according to claim 1, wherein the REP1 polypeptide has a sequence consisting of SEQ ID NO: 3. The method according to claim 1, wherein the polynucleotide sequence encoding the REP1 polypeptide has at least 70% identity with SEQ ID NO: 2. The vector according to claim 1, wherein the CBA promoter is constitutively active. The vector of claim 6, wherein the expression from the promoter is retinal-cell specific. The vector of claim 6, wherein the promoter further comprises a cytomegalovirus (CMV) enhancer. The vector of claim 1, comprising one or more additional regulatory sequences. 10. The vector of claim 9, wherein the one or more additional regulatory sequences comprise a postregulatory element (PRE). The vector according to claim 10, wherein the PRE comprises a woodchuck hepatitis postregulatory element (WPRE). The method according to claim 11, wherein the WPRE comprises a sequence having at least 70% identity with SEQ ID NO: 5. 12. The vector of claim 11, wherein the WPRE comprises or consists of SEQ ID NO: 5. The vector of claim 1, wherein the vector comprises 5′ AAV2 inverted terminal repeat (ITR) and 3′ AAV2 ITR. 15. The vector of claim 14, wherein the 5'AAV2 ITR comprises or consists of SEQ ID NO: 8. 15. The vector of claim 14, wherein the 3'AAV2 ITR comprises or consists of SEQ ID NO: 9. The vector of claim 1, wherein the vector is in the form of vector particles. The vector of claim 17, wherein the vector particle is serotype AAV2. A polynucleotide sequence encoding a REP1 polypeptide having at least 97% identity with SEQ ID NO: 3, the polynucleotide comprising an adeno-associated virus (AAV) vector operably linked to a chicken beta actin (CBA) promoter As a composition,
The composition comprises 1 x 10 ¹⁰ to 1 x 10 ¹³ genomic particles (gp/ml) of the AAV vector per milliliter. The composition of claim 19, wherein the composition comprises 1.0×10 ¹¹ gp/ml of the AAV vector. The composition of claim 19, wherein the vector further comprises the sequence of the AAV genome, and the AAV genome is a naturally-derived serotype or isolate or lineage of AAV. The composition of claim 21, wherein the serotype is AAV serotype 2 (AAV2). The composition of claim 21, wherein the sequence of the AAV genome comprises ITR. 24. The composition of claim 23, wherein the ITR comprises AAV2 ITR. The composition of claim 24, wherein the AAV2 ITR comprises or consists of the sequence of SEQ ID NO: 8 or SEQ ID NO: 9. The composition of claim 19, wherein the vector particle comprises one or more additional regulatory sequences. The composition of claim 26, wherein the additional regulatory sequence is a Woodchuck hepatitis post-regulatory element (WPRE). The composition of claim 27, wherein the WPRE comprises or consists of the sequence of SEQ ID NO: 5. The composition of claim 19, wherein the vector particle is serotype AAV2. A pharmaceutical composition for treating or preventing choroideremia in a patient in need thereof, comprising a therapeutically effective amount of a vector or composition according to any one of claims 1 to 29, wherein the vector is directly The composition is administered by retinal injection, subretinal injection or intravitreal injection. 31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition comprises or consists of 1Х10 ¹⁰ to 1Х10 ¹³ genomic particles (gp) per millimeter (mL), including an endpoint. 31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition comprises or consists of 1x10 ¹⁰ , 1x10 ¹¹ , 1x10 ¹² or 1x10 ¹³ gp per mL. 31. The pharmaceutical composition of claim 30, further comprising a liquid carrier. 31. The pharmaceutical composition of claim 30, wherein the vector is at a concentration of 1x10 ¹² gp per mL, the pharmaceutical composition further comprises a liquid carrier, and the pharmaceutical composition is administered directly into the subretinal space.

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